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Proteomics Based Analysis of the Nicotine Catabolism In www.nature.com/scientificreports OPEN Proteomics based analysis of the nicotine catabolism in Paenarthrobacter nicotinovorans Received: 11 February 2018 Accepted: 24 October 2018 pAO1 Published: xx xx xxxx Marius Mihăşan 1,2, Cornelia Babii1, Roshanak Aslebagh2, Devika Channaveerappa2, Emmalyn Dupree2 & Costel C. Darie2 Paenarthrobacter nicotinovorans is a nicotine-degrading microorganism that shows a promising biotechnological potential for the production of compounds with industrial and pharmaceutical importance. Its ability to use nicotine was linked to the presence of the catabolic megaplasmid pAO1. Although extensive work has been performed on the molecular biology of nicotine degradation in this bacterium, only half of the genes putatively involved have been experimentally linked to nicotine. In the current approach, we used nanoLC–MS/MS to identify a total of 801 proteins grouped in 511 non- redundant protein clusters when P. nicotinovorans was grown on citrate, nicotine and nicotine and citrate as the only carbon sources. The diferences in protein abundance showed that deamination is preferred when citrate is present. Several putative genes from the pAO1 megaplasmid have been shown to have a nicotine-dependent expression, including a hypothetical polyketide cyclase. We hypothesize that the enzyme would hydrolyze the N1-C6 bond from the pyridine ring with the formation of α-keto- glutaramate. Two chromosomally-encoded proteins, a malate dehydrogenase, and a D-3- phosphoglycerate dehydrogenase were shown to be strongly up-regulated when nicotine was the sole carbon source and could be related to the production the α-keto-glutarate. The data have been deposited to the ProteomeXchange with identifer PXD008756. Nicotine is the main alkaloid produced by the tobacco plant as an anti-herbivore chemical. Due to its potent parasympathomimetic stimulant efect, nicotine plays a crucial role in smoking addiction and proved to be toxic in high concentrations to both animals and humans1. Nicotine can accumulate as both liquid and solid tobacco waste during the manufacturing of tobacco products. As it is water soluble, nicotine can easily contaminate the environment and endanger human health and aquatic life. Several bacterial strains have been shown to use nicotine as their sole carbon and nitrogen source for growth. Generally referred as Nicotine-Degrading Microorganisms (NDMs)2, these bacteria offer an eco-friendly method for removing nicotine from tobacco products and waste by means of bioremediation. The NDMs could also provide enzymes and pathways that can be engineered to convert nicotine and other pyridine and/ or pyrrolidine ring containing compounds into starting materials for the synthesis of products of industrial and pharmaceutical importance. Te development of technologies for converting nicotine waste into 6-Hydroxy- 3-succinoyl-pyridine3, 3-succinoyl-pyridine4 or 6-hydroxy-nicotine5 as well as the recent identification of 6-hydroxy-nicotine as a neuroprotective drug6 shows that NDMs nicotine pathways and enzymes have valuable biotechnological applications and could be used for the production of green chemicals. Paenarthrobacter nicotinovorans is a soil Gram-positive NDM that frst came into the limelight in the 1960’s under the name Arthrobacter oxidans7, re-classifed as Arthrobacter nicotinovorans8 in the 1990’s and received its current name in 20169. P. nicotinovorans ability to use nicotine as carbon and energy source is linked to the presence of a cluster of nicotine-inducible genes (nic- genes) placed on the catabolic megaplasmid pAO110. P. nicotinovorans makes use of the pyridine pathway to degrade nicotine. Briefy, nicotine catabolism starts with 1Biochemistry and Molecular Biology Laboratory, Department of Biology, Alexandru Ioan Cuza University of Iaşi, Iaşi, Romania. 2Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY, USA. Correspondence and requests for materials should be addressed to M.M. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:16239 | DOI:10.1038/s41598-018-34687-y 1 www.nature.com/scientificreports/ hydroxylation of the pyridine ring and results in formation of γ-N-methylaminobutyrate (CH3-4-GABA) and 2,6-dihydroxy-pyridine (2,6-DHP). CH3-4-GABA is degraded to succinate and methylamine in what is called the lower nicotine pathway, the latter compound been shown to accumulate into the growth medium11. 2,6-DHP is hydroxylated to trihydroxy- pyridine (THP) which spontaneously dimerizes to 4,4′,5,5′-tetrahydroxy-3,3′-diaz adiphenoquinone-(2,2′) (Nicotine-blue, NB) giving the characteristic nicotine-blue color (for an overview see reference12, Fig. 1 and supplementary Fig. 1). Te remarkable work performed by the Decker and later the Brandsch group on the molecular biology and biochemistry of nicotine degradation in P. nicotinovorans13 lead to the experimentally establishment of func- tions for 19 out of the 40 genes making out the nic-gene cluster12. Te rest of the nic genes either have putative or no known functions or are may not be expressed at all. Tese genes might include some missing key points in nicotine metabolism such as major gene regulators or nicotine transporters. An experimental indication of the involvement of these putative genes in the nicotine metabolism is required. Te current shotgun proteomics approach attempts to fll this gap by identifying the proteins expressed by P. nicotinovorans in the presence of nicotine using nanoLC–MS/MS. A total of 801 proteins grouped in 511 non-redundant protein clusters were identifed when P. nicotinovorans was grown on citrate, nicotine and citrate, and nicotine as the only carbon source. Te diferences in protein expression patterns allowed us not only to fll in some of the blank spots in nicotine metabolism, but also to relate this plasmid-encoded catabolic pathway to the general chromosome-encoded pathways of the cell. Tis proteomics data can provide some valuable insight into the genetics and physiology of nicotine metabolism and serve as a basis for generating hypotheses for future attempts to genetically-engineer the catabolic pathway for increased bioremediation efciency or production of green chemicals. Results and Discussion Proteins identifcation overview. Te cell free lysates from Paenarthrobacter nicotinovorans pOA1 grow- ing on citrate, nicotine and citrate and nicotine as the only carbon sources were frst separated by sodium dodecyl sulfate-polyacrylamine gel elctrophoresis (SDS-PAGE), then digested with trypsin and the resulting peptides ana- lyzed by nanoLiquid Chromatography Tandem Mass Spectrometry (nanoLC-MS/MS). A bottom-up approach based on the MASCOT MS/MS database search algorithm was used to identify the proteins in each sample. Currently, there is no fnal genome of P. nicotinovorans pAO1 available, so for the database searches a custom database was used. Te database included the reference proteome of P. aurescens strain TC1, the complete pro- teome of pAO1 megaplasmid as well as the partial proteome derived from the draf genome of P. nicotinovorans strain Hce-1 (WGS sequence set BDDW01000000). Tis approach allowed us to identify a total of 801 proteins grouped in 511 non-redundant protein clusters. Te full list of proteins identifed in this study is presented in Supplementary Table 1. Te distribution of these clusters showing the overlap between the proteomes of P. aureus TC1 and P. nicotinovorans is depicted in Table 1. A total number of 368 non-redundant proteins were identifed based on genes present in both the P. aureus TC1 and the P. nicotinovorans making out the core-genome of the two species. Moreover, a number 115 non-redundant proteins hits were identifed based on genes that are only found in the P. nicotinovorans pangenome, 28 being encoded by the pAO1 megaplasmid. One unexpected fnding is that a number of 30 non-redundant proteins hits were identifed based on genes that are specifc for P. aureus TC1 genome. Tis can only be explained by the fact that the WGS sequence set BDDW01000000 is not the com- plete genes set for the P. nicotinovorans strain. Te overlap between the shared and unique proteins expressed in P. nicotinovorans pAO1 cells growing on dif- ferent carbon sources is summarized in the Venn diagram from Fig. 2. Disregarding whether it is used as the sole Carbon source, the presence of nicotine in the growth medium triggers several important changes in the protein expression patterns of P. nicotinovorans. Te magnitude and signifcance of these changes can be observed in the Volcano plots depicted in Fig. 3. Due to the specifc way the Scafold sofware calculates the fold change, the pro- teins not expressed in any of the compared conditions do not show up on the graphs. In the cells grown solely on nicotine, 46 out of 632 identifed proteins were dysregulated when compared to the citrate grown nicotine cells. When both nicotine and citrate were present as the carbon source, the number of dysregulated proteins was only 16 out of 618. Te full list of statistically signifcant dysregulated proteins in all growth conditions is presented in Supplementary Table 2. Diferentially regulated plasmid-encoded proteins in P. nicotinovorans in the presence of nicotine. As expected, none of the proteins encoded by the pAO1 megaplasmid and known to be involved in nicotine metabolism could be identifed in the cell free extracts from the cells grown on citrate alone. Te presence of nicotine in the growth media alone or in combination with citrate
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